It is estimated that alcohol dependency affects over 10 million Americans and that the cost of alcohol related problems totals over 100 billion dollars. Alcohol consumption impairs both humoral and cell-mediated immunity. Natural killer (NK) cells play an important role in immunosurveillance against certain infectious diseases and cancer and are especially active in preventing tumor metastasis. NK cells also control the rate of progression of aids. We showed previously that alcohol consumption specifically results in impaired NK and lymphokine activated killer (LAK) cytolytic activity when mice consume between 25-40% of their calories from ethanol administered in the drinking water. The objective of this proposal is to determine the mechanism(s) underlying this impaired cytolytic activity and to specifically test the hypothesis that alcohol consumption impairs cytolytic activity through disruption of protein kinase C cell signaling, which in turn modulates granule and cytotoxic mediator function. We will examine the effect that alcohol consumption has on NK granule cytotoxicity and the specific function of the cytolytic mediators, perforin, granzyme A, and granzyme B. We will also determine the effect of alcohol consumption on the activity and amount of these proteins in the two primary subpopulations of NK and LAK cells that contribute to inherent NK cell cytolytic activity (NK1.1+LGL1-cells) and to LAK cytolytic activity (NK1.1+LGL-cells). In vitro colorimetric enzymatic assays will characterize granzyme A and granzyme B proteolytic activity. Isolation of enriched NK1.1+ cells and the LGL1 subpopulations will utilize magnetic bead separation technology. Western blot analysis will be used to determine protein expression in granzymes, and reverse transcriptase polymerase chain reaction (RT-PCR) will be used to determine mRNA. Protein kinase C activity will be determined using standard assays. Cytolytic granules will be isolated from NK/LAK cells with nitrogen cavitation.